Langmuir 2000, 16, 8217-8220
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Charge Displacement by Adhesion and Spreading of a Cell: Amperometric Signals of Living Cells Vesna Svetlicˇic´,* Nadica Ivosˇevic´, Solveg Kovacˇ, and Vera Zˇ utic´ Center for Marine and Environmental Research, Rudjer Bosˇ kovic´ Institute, P.O. Box 180, 10002 Zagreb, Croatia Received June 5, 2000. In Final Form: August 1, 2000
Introduction The electrochemical techniques employing microelectrodes and high-sensitivity time-resolved recording of amperometric signals1 allow detection in real time of single eventssoxidation or reduction of nano- and microparticles. In contrast to traditional electrochemistry of solutes, where solution transport, adsorption, and electrode kinetics control the response, in electrochemistry of microparticles2 either the particles are already immobilized on the electrode surface or the initial attachment of a free particle is established by an interplay of hydrodynamic and surface forces.3 Random appearance of amperometric signals has been recorded in inorganic suspensions using a mercury microdrop electrode,4 as well as in biological systems using carbon-fiber microelectrodes.5-10 The random appearance of single events is due either to the spatial heterogeneity inherent to dispersed systems or to a stochastic nature of neurotransmitter secretion by exocytosis.7 In both cases the information on single events was obtained through analysis of the spike-shaped signals. The spike area, expressed in units of charge Q, was related by Faraday’s law to the size of a colloidal particle4 or to a vesicle radius.5 There is, however, an intriguing ambiguity in interpretations of single events corresponding to vesicular secretion by exocytosis. The signal shape could not be fitted by diffusion models10 and an additional process, on a time scale of milliseconds,8 appeared to be rate-controlling. Sluyters and co-workers4 have clearly demonstrated that in case of Hg(I) colloids there is a narrow potential range where reduction current spikes appeared, compared to the broad potential range of mercury(I) reduction. The authors offered an explanation in terms of electrostatic repulsion between electrode double layer and colloidal particles. They also noticed a correlation with the phe* To whom correspondence may be addressed. Phone: 385 1 4561 185. Fax: 385 1 4680 242. E-mail:
[email protected]. (1) Ryan, M. D.; Bowden, E. F.; Chambers, J. Q. Anal. Chem. 1994, 66, 360R. (2) Scholz, F.; Meyer B. In Electroanalytical Chemistry: A Series of Advances; Bard, A. J., Rubinstein, I., Eds.; M. Dekker: New York, 1998; pp 1-86. (3) Mackay, R. A.; Texter, J. Electrochemistry in Colloids and Dispersions; VCH Publishers: New York, 1992. (4) Baars, A.; Sluyters-Rehbach, M.; Sluyters, J. H. J. Electroanal. Chem. 1990, 283, 99. (b) Baars, A.; Sluyters-Rehbach, M.; Sluyters, J. H. J. Electroanal. Chem. 1994, 368, 11. (5) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawaagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, Jr., E. J.; Viveros, O. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 110754. (6) Schroeder, T. J.; Jankowski, J. A.; Kawagoe, K. T.; Wightman, R. M. Anal. Chem. 1992, 64, 3077. (7) Chow, R. H.; von Ru¨den, L.; Neher, E. Nature 1992, 356, 60. (8) Jankowski, J. A.; Schroeder, T. J.; Ciolkowski, E. L.; Wightman, R. M. J. Biol. Chem. 1993, 268, 14694. (9) Kennedy, R. T.; Huang, L.; Atkinson, M. A.; Dush, P. Anal. Chem. 1993, 65, 1882. (10) Schroeder, T. J.; Jankowski, J. A.; Senyshyn, J.; Holz, R. W.; Wightman, R. M. J. Biol. Chem. 1994, 269, 17215.
Figure 1. Schematic diagram of attractive interaction of a cell with a positively charged mercury electrode in an aqueous electrolyte solution. The current-time transient (adhesion signal) is caused by the double-layer charge displacement from the contact area AC.
nomenon of charging current spikes discovered at the dropping mercury electrode immersed in dispersion of electroinactive oil droplets.11 The charging current spikes could be recorded at the positively and negatively charged electrode, but only within the potential range limited by critical interfacial tension at the three-phase boundary mercury electrode/aqueous electrolyte/organic liquid, defined according a modified Young-Dupre´ equation.11 These results indicate a more general significance of adhesion phenomena in single particle-electrode interaction. The aim of the present work is to trace adhesion response of single particles without influence of electron redox exchange at the electrode. Cells with fluid or a flexible outer membrane can readily form adhesive contact with the substrate with little or no resistance to oppose deformation.12,13 We selected living cells of unicellular marine algae as a model soft particle and the mercury electrode as adhesion sensor. Attractive interaction between a cell and electrode should result in a double layer charge displacement, as represented by a simplified scheme in Figure 1. The charge displacement causes a flow of compensating current, ID, that is directly related to the formation of adhesion contact:
dA σ dt E
ID ) -
(1)
A is the area of the contact interface, t is time, and σE is the surface charge density of the electrode/aqueous electrolyte interface. The amount of displaced charge, qD, is determined from the adhesion signal (Figure 1):
qD )
∫tt +τ i dt 1
1
(2)
The area of the contact interface formed, AC, can then be determined with precision, surface charge of the electrode being known:
AC ) qD/σE
(3)
Dunaliella tertiolecta cells (Figure 2) are suitable for (11) Zˇ utic´, V.; Kovacˇ, S.; Tomaic´, J.; Svetlicˇic´, V. J. Electroanal. Chem. 1993, 349, 173. (12) Evans, E. In Physical Basis of Cell-Cell Adhesion; Bongrand, P. Ed.; CRC Press, Inc.: Boca Raton, FL, 1988; pp 91-123. (13) Seifert, U. Adv. Phys. 1997, 46, 13.
10.1021/la0007832 CCC: $19.00 © 2000 American Chemical Society Published on Web 09/26/2000
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Notes
Figure 3. (a) Current-time curve at the positively charged electrode (E ) -400 mV, σHg ) +3.8 µC/cm2) recorded in a deairated cell suspension of 5.4 × 105 cell/mL in 0.1 M NaCl. Adhesion signals appear as spikes superimposed on a flat background current. (b) Current-time curve at the negatively charged electrode (E ) -800 mV, σHg ) -6.5 µC/cm2) at otherwise identical conditions as in Figure 3a.
Figure 2. Electron micrograph of thin section through D. tertiolecta cell, showing the cell membrane and details of cell structure; magnification 18000×, bar denotes 1 µm (courtesy of Dr. Mercedes Wrischer).
electrochemical detection because of their size, membrane properties,14 and euryhaline nature (growing over a wide salinity range). The cells are simple to grow in the laboratory in axenic (uncontaminated) culture. In a variety of aqueous electrolyte solutions they form stable suspensions of single cells due to their pronounced motility and low stickiness. We use a fast dropping mercury electrode because of its well-defined interfacial properties and renewable surface.15,16
Electrochemical Measurements. The electrochemical experiments were performed in a standard Methrom vessel with a 20-mL volume of cell suspension. A fast dropping mercury electrode with drop life 2.0 s, flow rate 6.03 mg/s, and maximum surface area 4.57 mm2 was used with a 0.1 M Ag/AgCl reference electrode in a three-electrode configuration. The amperometric measurements were conducted after careful deairation of aqueous electrolyte solution by a stream of nitrogen in order to eliminate any interference of the faradaic current of oxygen reduction. The aliquot of stock cell suspension was added directly into the deairated solution, prior to electrochemical measurement. The aqueous solution contained 0.1 M NaCl and 5 mM NaHCO3 to maintain pH at 8.2. Water used for preparation of the solutions was ultrapure MilliQ water. Measurements were performed at 20 and 25 °C. A PAR 174A polarographic analyzer was used for measurements of current-time transients at a constant potential. The fast transients were recorded and stored using a Nicolet 3091 digital oscilloscope with time resolution of 20 µs per point for recording single adhesion signals and 500 µs per point for recording current-time curve over a whole drop lifetime (2 s). At least 30 current-time curves were recorded at a given constant potential. All potentials are referred to the Ag/AgCl electrode. Data for the interfacial tension (γ12) and surface charge density of the mercury electrode (σHg) were taken from literature.18
Experimental Section Cell Cultures and Cell Suspensions. We used a laboratory culture of unicellular marine algae Dunaliella tertiolecta Butcher (Chlorophyceae). The cells (maximum dimension 6-12 µm) were grown in seawater enriched with F-2 medium17 in batch culture. Cell density in culture reached up to 2 × 106 cells/mL after 8 days of growth. The cells were separated from the growth medium with mild centrifugation (1500g, 5 min). The loose pellet was washed several times and resuspended in filtered seawater to form a stock suspension containing 107 cells/mL. Suspensions submitted to electrochemical measurements (104-6 × 105 cells/ mL) were controlled by light microscopy in terms of cell densities and viability. (14) Jokela Tang Chung-Chau, A. Outer Membrane of Dunaliella tertiolecta: Isolation and Properties. Ph.D. Thesis, University of California, San Diego, 1969. (15) Heyrovsky´, J.; Ku˚ta, J. Principles of Polarography; Publishing House of Czechoslovak Acadaemy of Science: Prague, 1965. (16) Levich, V. G. Physicochemical Hydrodymamics; Prentice Hall: Engelwood Cliffs, NJ, 1962. (17) Guillard, R. R. In Culture of marine Invertebratae animals; Smith W. L., Chanley, M. H., Eds.; Plenum Press: New York, 1975; p 22.
Results The electrochemical response in a cell suspension is illustrated in Figure 3a by a segment of a current-time curve where the sequence of adhesion signals appeared. The curve was recorded at the potential -400 mV, where mercury surface is positively charged, σHg) +3.8 µC/cm2. The signals are characterized by a steep rising portion followed by a slower decay of the displacement current. Subsequent signals on the same i-t curve do not seem to influence one another. The signals differ only slightly in the peak current and duration, indicating attachments from a nearly monodisperse particle population. The similarity of signals shape and duration to those of droplets of insoluble electroinactive organic liquids11 implies that the underlying mechanism of cell adhesion should involve (18) Graham, D. C. J. Am. Chem. Soc. 1949, 71, 2975. (b) Lyklema, J.; Parsons, R. In Compilation of Data on the Electrical Double Layer on Mercury Electrodes; Office of Standard Reference Data, National Bureau of Standards, Department of Commerce: Washington, DC, 1983.
Notes
Langmuir, Vol. 16, No. 21, 2000 8219 Table 1. Analysis of Adhesion Signals of D. Tertiolecta Cellsa current, iP/µA
duration, τ/ms
displaced charge, qD/nC
contact area, AC/×104 cm2
1.3 1.4 1.2 1.2 1.2 1.15 2.2 1.6 1.35 1.35 1.65 1.55
4.4 6.0 4.0 5.2 4.4 4.4 5.6 5.2 4.4 5.2 6.8 5.2
0.7 1.2 1.0 1.0 1.0 0.95 1.1 1.2 0.9 1.4 1.4 1.3
1.84 3.16 2.63 2.63 2.63 2.52 2.89 3.16 2.37 3.68 3.68 3.42
a Signals were recorded in suspension of 5.4 × 105 cells/mL in 0.1 M NaCl at a potential of -400 mV. Initial surface charge density of the mercury electrode is +3.8 µC cm-2.
Figure 4. Dependence of adhesion signal frequency of D. tertiolecta cells on applied potential in oxygen-free cell suspension (0.1 M NaCl, cell density 2.7 × 105 mL-1). Ten consecutive current-time curves were analyzed, and bars denote standard deviation. (insert) Plot of surface charge density absolute values, |σHg| (0) and interfacial tension at DME, γ12 (4), vs applied potential in 0.1 M NaCl.
the same sequence of events: fast initial attachment and deformation followed by a slower spreading forming the contact interface of a finite area (Figure 1). If the current spikes in Figure 3a originate from the double-layer charge displacement, they should disappear completely at the potential of zero charge (Epzc), while at the negatively charged electrode (E < Epzc) the amperometric signals should be reversed.11 Indeed, signals could not be detected at Epzc. The signals recorded at a negatively charged electrode (-800 mV, Figure 3b) appear as mirror images of the signals in Figure 3a. This also proves that the distance of the closest approach of a cell, at a positively and negatively charged electrode, is equal or less than the distance of the plane going through the charge centers of hydrated counterions nearest to the electrode.19,20 It is interesting to point out the absence of any detectable redox current which may originate from possible redox reactions of electroactive substances located on the cell surface. We further analyzed suspensions with increasing cell density in the range from 104 to 6 × 105 cells/mL. At each constant potential we recorded series of 30 i-t curves on consecutive mercury drops because of the stochastic nature of the cell encounters with the electrode.21 The average number of adhesion signals per one drop life, expressed as the attachment frequency, is proportional to cell concentration. We further looked for adhesion signals in a broader range of applied potentials, from 0 to -1.8 V. The adhesion signals appeared only in the potential range between -85 and -920 mV. At the potentials more positive than -85 mV and more negative than -920 mV the cells behave as inert particles. Within the potential range of adhesion the signal frequency depends strongly on the potential (Figure 4). Signal frequency reaches maximum values at the potentials between -300 and -400 mV, where interfacial tension of DME is close to its maximum value and hydrophobic interaction is expected to prevail in cell (19) Bockris, J. O’M.; Reddy, A. K. N. Modern Electrochemistry; A Plenum/Rosetta Edition: London, 1973; Vol. 2, pp 718-801. (20) Marcˇelja, S. Nature 1997, 385, 689. (21) Kovacˇ, S.; Kraus, R.; Gecˇek, S.; Zˇ utic´, V. Croat. Chem. Acta 2000, 73, 279
adhesion. The insert in Figure 4 shows the corresponding surface charge densities and interfacial tension of DME. At the potential of zero charge of DME, Epzc, the signal frequency drops to zero since there is no surface charge to be displaced. The critical potential of adhesion at the positively charged DME corresponds to γ12 ) 394 mJ/m2 and at the negatively charged DME to 407 mJ/m2 (evaluated from the electrocapillary data18). The difference in critical γ12 values11,22 of D. tertiolecta could serve as a measure of electrostatic interactions since the cell exterior is negatively charged. The exact value of the surface charge of D. tertiolecta living cells could not be determined by electrophoresis due to active movements of the cells. Examples of signal analysis in terms of peak current (ip), duration (τ), displacement charge (qD), and final area of the contact interface (AC) at the positively charged electrode is given in Table 1. qD was obtained by graphical integration of adhesion signals, and AC was calculated according to the eq 3. The size distribution of cell contact areas is (1.84-4.2) × 10-4 cm2. The variation in AC values can be ascribed to the distribution of cell sizes in the culture. The contact interface area, AC, exceeds crosssection area of a free cell by 2 orders of magnitude. Evidently, the D. tertiolecta cell ruptured during the spreading process. It is known for vesicles that strong adhesion always leads to vesicle rupture.13 The recent experiments performed using atomic force microscopy23,24 allowed direct visualization of vesicle adhesion at the mica/water interface, with a nanometer vertical and lateral resolution. Immediately the vesicle adhered on the mica surface, it ruptured spontaneously, and deformed to a flat supported bilayer or “single bilayer disk”24 on the mica. Conclusion We have presented the amperometric response of single cell adhesion in real time, from the initial attachment to a finite state of spread cell at the growing mercury drop electrode. The technique is based on measurement of double-layer charge displacement. The only hypothesis used in interpreting the signals is the validity of the electrical double-layer model. The flow of compensating current reflects the dynamics of adhesive contact formation and subsequent spreading of a cell. The rate of adhesion and spreading of cells is enhanced by the hydrodynamic (22) Ivosˇevic´, N.; Zˇ utic´,V.; Tomaic´, J. Langmuir 1999, 15, 7063. (23) Egawa, H.; Furusawa, K. Langmuir 1999, 15, 1660. (24) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806.
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regime of an electrode’s growing fluid interface. The spikeshaped signals have the peak current in microampere range, duration of 5-10 ms, and displaced charge in the nanocoulomb range. The electrochemical technique thus allows a precise measurement of the contact area between the cell and the electrode. The distance of the closest approach of an adhered cell can be estimated with certainty as equal or smaller than the outer Helmholtz plane, i.e., 0.3-0.5 nm. There is clear evidence of cell rupture in the potential range of maximum attractive interaction, which is around the potential of zero charge. A surprising similarity to adhesion signals of droplets of liquid hydrocarbons (C12-C18)22 suggests that collective properties of cell exterior govern the dynamics of adhesion and rate of spreading, with fluidity playing a major role. Phospholipid vesicles, the classical model in studying physical mechanisms of cell adhesion,12,13 would be well suited for future electrochemical studies. Our results demonstrate a general significance of adhesion phenomena in a single particle-electrode interaction. When electroactive molecules reside in an
Notes
organized microenvironment, such as colloidal particles, microdroplets, or vesicles, their redox reaction at the electrode is preceded by adhesion step that is likely to become rate-determining. The characteristic potential range of adhesion can serve to study the interplay of complex surface forces25 involved in soft particle-electrode double-layer interactions.26,27 Acknowledgment. We thank Dr. John Green from the Plymouth Marine Laboratory for the Dunaliella terciolecta culture. The research was financially supported by the Ministry of Science and Technology of the Republic of Croatia, Project P-1508. LA0007832 (25) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1991. (26) Horn, R. G.; Bachmann, D. J.; Connor, J. N.; Miklavcic, S. J. J. Phys.: Condens. Matter 1996, 8, 9483. (b) Bachmann, D. J.; Miklavcic, S. J. Langmuir 1996, 12, 4197. (27) Ivosˇevic´, N.; Zˇ utic´, V. Langmuir 1998, 14, 231.
